Sinorhizobium meliloti integration host factor (IHF) genes affect symbiotic performance of alfalfa ( Medicago sativa L.)

Integration host factor (IHF) is a histone-like bacterial priotein that can sequence-specifically bind to DNA [1]. IHF has diverse effects on gene expression, though these effects are usually not very strong. IHF is required for transcriptional activation of some δ 54 promoters in a number of Gram-negative bacteria by assisting transcription factors in forming a transcription-competent open promoter complex [2]. In nitrogen-fixing bacteria, transcriptional activation of nitrogen fixation operons (nifand fixgenes) usually requires δ 54 (an alternative δ factor) and the activator protein NifA [3]. For example, NifA-mediated transcriptional activation of the nifH promoter is stimulated by IHF in Klebsiellapneumoniae. Similarly, NifA-mediated transcriptional activation of the nifH and nifD promoters is strongly enhanced by IHF in Bradyrhizobium japonicum, whereas only a weak activation of the nifH promoter was reported for Sinorhizobium (Ensifer) meliloti [4]. It was suggested that IHF improves the efficiency and specificity of

Integration host factor (IHF) is a histone-like bacterial priotein that can sequence-specifically bind to DNA [1]. IHF has diverse effects on gene expression, though these effects are usually not very strong. IHF is required for transcriptional activation of some δ 54 promoters in a number of Gram-negative bacteria by assisting transcription factors in forming a transcription-competent open promoter complex [2]. In nitrogen-fixing bacteria, transcriptional activation of nitrogen fixation operons (nif and fix genes) usually requires δ 54 (an alternative δ factor) and the activator protein NifA [3]. For example, NifA-mediated transcriptional activation of the nifH promoter is stimulated by IHF in Klebsiella pneumoniae. Similarly, NifA-mediated transcriptional activation of the nifH and nifD promoters is strongly enhanced by IHF in Bradyrhizobium japonicum, whereas only a weak activation of the nifH promoter was reported for Sinorhizobium (Ensifer) meliloti [4]. It was suggested that IHF improves the efficiency and specificity of symbiotic expression of the fixABCX operon in rhizobia [5]. IHF can also stimulate the expression of genes of the rhizobial dicarboxylic acid transport (Dct) system. DctD (the response regulator)-mediated activation of the dctA promoter was reported for Rhizobium leguminosarum, whereas activation of dctA in S. meliloti was found to be independent of IHF in vitro [6].
S. meliloti infects legume host plants such as Medicago sativa L. (alfalfa) and M. truncatula Gaertn. to induce the formation of nitrogen-fixing root nodules. At the early stage of symbiosis, flavonoids such as luteolin secreted by host roots bind to the LysR family regulator protein NodD1. The flavonoid-NodD1 complex subsequently activates the expression of nodulation genes (such as nodABC) to synthesize bacterial nodulation signals known as Nod factors [7]. Medicago plants perceive Nod factors of S. meliloti by specific LysM-type receptor kinases to stimulate the expression of genes required for bacterial infection and nodule primordium development [8]. In formed nodules, bacteria are released from infection threads into host cells and differentiate into nitrogen-fixing bacteroids. Expression of the nitrogenase genes nifHDK and fixABC depends on NifA and also partially on IHF proteins encoded by ihfA and ihfB [5,6]. Whether S. meliloti IHF affects alfalfa nodulation at early symbiotic stages has not been investigated, however. In the present work, the symbiotic functions of the S. meliloti infA/B genes were analyzed in nodulation tests with constructed deletion mutants. Strains and plasmids are shown in Supplementary Table  S1 and primers are shown in Supplementary Table S2.
Three infA/B deletion mutants of S. meliloti Sm1021 (ΔihfA, ΔihfB and the double mutant ΔihfAB) were constructed using a suicide plasmid-homologous recombination strategy (Supplementary Figure S1A). The mutants with correct double-cross recombination event were identified by PCR (Supplementary Figure S1B,C), and the mutations were confirmed by DNA sequencing. Total RNA was extracted from free-living Sinorhizobium cells and reverse transcription PCR (RT-PCR) was performed to determine the transcript levels of infA/B. The infA/B transcripts could not be detected in the deletion mutants while considerable expression was found in the parent strain Sm1021 (Supplementary Figure S1D,E). These results confirmed that the infA/B deletion mutants were successfully constructed.
Growth curves with LB/MC broth were determined to evaluate the effects of infA/B on bacterial growth and proliferation. The results indicated that the logarithmic growth phase was delayed in the three mutants as compared to Sm1021 ( Figure 1A). These results suggest that ihfA and ihfB can affect growth of free-living S. meliloti bacteria under the tested condition.
Furthermore, cell motility assays using LB/MC swimming plates were performed with the constructed mutants. The results indicated that the three mutants swam obviously slower than Sm1021 ( Figure  1B and Supplementary Figure S2). These observations suggest that there is a genetic connection between ihfA/B genes and genes cluster involving in bacterial flagellum biosynthesis and motility.
To evaluate the symbiotic phenotypes of the ihfA/B deletion mutants, S. meliloti suspensions were inoculated onto roots of alfalfa seedlings. Plants were harvested four weeks later. Plants inoculated with the deletion mutants showed reduced growth when compared to those inoculated with Sm1021 ( Figure 1C). The shoot biomass (dry weight) of alfalfa plants inoculated with ihfA/B deletion mutants was approximately one third lower than that of plants inoculated with Sm1021 ( Figure 1D). As compared to parent strain Sm1021, the inoculation of three mutants resulted in the impaired nodule formation of host plant. Plants inoculated with the mutants formed fewer nodules both in total number and in pink ( Figure 1E,F). In general, pink nodule is thought of being normal in production of leghemoglobin that is essentially required for the nitrogen fixation process. Furthermore, we measured the nitrogen fixation capacity of the nodules by the acetylene reduction method. The results showed that the nitrogenase activity of nodules induced by the mutants was significantly decreased relative to that of Sm1021 (8.7, 7.3 and 2.1 nmol C 2 H 4 /mg nodule protein/min for ΔihfA, ΔihfB and ΔihfAB, respectively; 20.8 nmol C 2 H 4 /mg nodule protein/min for Sm1021) ( Figure 1G). Taken together, these data indicate that the ihfA/B genes are important for nitrogen fixation in alfalfa nodules.
To evaluate the effects of ihfA/B deletions on the bacterial nodulation competitiveness, Sm1021 and ΔihfAB bacteria were mixed at a 1:1 or 1:9 (Sm1021:ΔihfAB) ratio and then inoculated onto alfalfa seedlings. Nodule occupation ratios were determined three weeks after inoculation. The results showed that all of the examined nodules contained Sm1021 at the 1:1 inoculation ratio and that 99% of the nodules were occupied by Sm1021 bacteria at the 1:9 inoculation ratio. These findings show that the ihfA and ihfB genes promote the establishment of symbiosis in competitive nodulation experiments.
Since IHF interacts with RNA polymerase and transcription factors to regulate transcription, it can be expected that the observed symbiotic phenotype of the ihfA/B deletion mutants are resulted from differential gene expression. To test this possibility, total RNA from free-living S. meliloti cells in LB/MC cultures was extracted for whole genome RNA-seq analysis. As shown in Supplementary Table S3 (q value <0.05 and FC2), 1026, 476 and 1036 genes were found to be differentially expressed in ΔihfA, ΔihfB and ΔihfAB relative to the parent strain Sm1021. The number of significantly upregulated genes was 336, 117 and 206, whereas that of significantly downregulation genes was 690, 359 and 830 respectively in these mutants. Remarkably, the three mutants showed downregulation of chemotaxis and flagellum biosynthesis genes to a varying degree (Figure 2A). Downregulation of five selected genes (possibly involved in nodulation competitiveness) was verified by quantitative RT-PCR ( Figure 2B). Furthermore, the RNA-seq data indicated a downregulation trend of nodulation genes in the mutants ( Figure 2C). Quantitative RT-PCR confirmed the downregulation of key nodulation genes in the ΔihfAB mutant. Expressions of nodC and nodD1 in the mutant were also lower than those in Sm1021 when cells were treated with a flavonoidcontaining extract from alfalfa seeds ( Figure 2D). These results indicate that ihf genes of S. meliloti are important for transcriptional activation of key nodulation genes, suggesting that they affect the synthesis of Nod factors. To evaluate the activity of Nod factors produced by the mutants, quantitative RT-PCR was used to determine the transcript levels of the early nodulin gene MtENOD11 of M. truncatula, whose expression is known to be rapidly induced by Nod factors [9]. The results showed that the cell suspensions of the ihfA/B mutants inefficiently induced the expression of MtENOD11 in M. truncatula seedlings when compared to the suspension of Sm1021 or a treatment with purified NodSm-IV (C16:2, S), a tetrameric Nod factor of S. meliloti ( Figure 2E). These results indicate that ihfA/B are regulator genes of Nod factor synthesis. Thus, impaired nodule formation of alfalfa inoculated with ihfA/B mutants ( Figure 1D,E) could be due to the downregulation of nodulation genes and reduced Nod factor production.
As alfalfa nodules induced by ihfA/B deletion mutants showed reduced nitrogen fixation ( Figure 1G), we suggest that the lack of ihf genes also affects the expressions of nitrogen fixation (nif or fix) genes as proposed in a published report [10]. We therefore extracted total RNA from four-week-old alfalfa nodules and performed quantitative RT-PCR for representative nif and fix genes. The results indicated that nifA, nifD, nifH and fixT transcripts were reduced in nodules induced by the ihfA/B mutants when compared to nodules harboring Sm1021 (Supplementary Figure S3). These results are consistent with previous work which reported similar nif and fix gene expression data in other rhizobial strains [5,6].
In conclusion, the free-living and symbiotic phenotypes of constructed S. meliloti ihfA/B deletion mutants showed altered growth and motility properties, reduced the capacity to form effective nitorgen-fixing nodules on alfalfa. Accordingly, expression of nif and fix genes was reduced in mature nodules. Remarkably, we also found that ihfA/B deficient mutants show downregulation of nod genes such as nodD1 and nodC which are required for Nod factor production and infection of host legumes. Hence, the ihfA and ihfB of S. meliloti can be considered as global transcriptional regulator genes that affect the nodule symbiosis at early and late symbiotic stages.

Supplementary Data
Supplementary data is available at Acta Biochimica et Biophysica Sinica online.

Funding
This work was supported by the grant from the Natural Science Foundation of China (No. 31370277 to L.L.).